Fluorescent labeling reagents have become increasingly useful investigative tools. The wider use of fluorescently labeled probes has resulted partly from advances in instrumentation and partly from the availability of new and improved fluorescent dyes. The cyanine dyes have received particular interest since relatively minor alterations in their chemical structure allows for variation in their excitation and emission wavelengths, an advantage for designing multicolor systems useful for simultaneous detection of more than one fluorescent probe.
More recently the cyanine dyes have become widely used as one component of a tandem conjugate with a second fluor, often proteinaceous fluorophores such as phycoerythrin (PE) or Peridinin-chlorophyll a-protein (PerCP). When the emission spectrum of one fluor overlaps the excitation spectrum of another, and they are sufficiently close to each other (&lt;10 nm), it is possible for the excitation energy of the first fluor to be transferred to the second through a fluorescent resonance energy transfer process (Glazer and Stryer, Biophys J. 43:383-386, 1983).
A significant drawback to the use of these reagents, particularly for labeling antibodies used in analysis and sorting of blood cells, is the tendency for the fluor complex to bind to components of the system in an indiscriminate manner (van Vugt, et al, Blood 88:2358-2359, 1996; Beavis and Pennline, Cytometry 24:390-394, 1996; Shapiro, Practical Flow Cytometry, 3rd ed, pg 282, 1995).
A need, therefore, still exists for new labeling reagents that are sensitive, easily detected, and exhibit little or no undesirable fluor-mediated binding.